Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is a vitally important molecule in eukaryotic signal transduction pathways, involved in the control of cell proliferation, differentiation, and oncogenic transformation. PtdIns(4,5)P2 is hydrolyzed by phospholipase C (PLC) to produce the second messengers inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (DG). Ins(1,4,5)P3 triggers release of calcium from internal stores and thus modulates Ca2+ -regulated pathways, while DG activates the phospholipid- and Ca2+ -dependent protein kinase C. PtdIns(4,5)P2 itself regulates other important functions, such as the activity of several actin binding proteins. Clearly, PtdIns(4,5)P2's intracellular concentration must be stringently regulated, but almost nothing is known about this regulation. The broad, long term objective is to elucidate the mechanisms regulating the homeostasis of PtdIns(4,5)P2 and to determine the role of this regulation in signal transduction. The specific aims of this proposal employ the two-hybrid screen and the powerful experimental advantages of the yeast Saccharomyces cerevisiae to identify proteins which interact with yeast phospholipase C (Plclp) and to determine the significance of the interaction(s) for the in vivo function of the enzyme. The applicant has performed the two-hybrid screen and isolated many plasmids which fall into at least 6 different groups. The applicant will complete this work and will identify the corresponding genes by sequencing (SA1). The plan is to confirm the interaction between Plclp and proteins encoded by these genes by independent biochemical techniques (SA2). The intent is to identify the domain(s) of Plclp responsible for the interactions (SA3) and to determine the roles of these interactions for the in vivo functions of Plclp (SA4).